Applied and Environmental Microbiology最新文献

Mammalian alkaline phosphatase (AP) is widely used in diagnostics and molecular biology but its widespread use is impaired because it is difficult to express in Escherichia coli and has low thermostability. To overcome these challenges, we employed sequence-based protein redesign methods, specifically full consensus design (FCD) and ancestral sequence reconstruction (ASR), to create APs with enhanced properties. Biochemical analyses revealed that these newly designed APs exhibited improved levels of expression in their active form and increased thermostability compared to bovine intestinal AP isozyme II (bIAPII), without impeding enzymatic activity. Notably, the activity in culture broth of the designed APs was an order of magnitude higher than that of bIAPII, and their thermal stability increased by 13°C-17°C (measured as T₅₀). We also assessed 28 single-point mutants of bIAPII to identify regions influencing thermostability and expression level; these mutations were common in the engineered APs but not in bIAPII. Specific mutations, such as T413E and G402S, enhanced thermostability, whereas increasing the expression level required multiple mutations. This suggests that a synergistic effect is required to enhance the expression level. Mutations enhancing thermostability were located in the crown domain, while those improving expression were close to the dimer interface, indicating distinct mechanisms underpinning these enhancements.

Importance: Sequence-based protein redesign methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR), have the potential to construct new enzymes utilizing protein sequence data registered in a rapidly expanding sequence database. The high thermostability of these enzymes would expand their application in diagnostics and molecular biology. These enzymes have also demonstrated a high level of active expression by Escherichia coli. These characteristics make these APs attractive candidates for industrial application. In addition, different amino acid residues are primarily responsible for thermal stability and active expression, suggesting important implications for strategies for designing enzymes by FCD and ASR.

As a response regulator of the EsrA-EsrB two-component system, EsrB is conserved in Hafniaceae and plays a crucial role in virulence and pathogenicity. EsrB possesses DNA binding abilities, enabling it to regulate the transcription of virulence genes to confront different stresses and achieve systematic infections. Here, ChIP-seq analysis of EsrB in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments) revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7'' pseudopalindromic DNA motif and interact with metabolic-related promoters with a high AT DNA motif. The crystal structure of the C-terminal of EsrB (EsrB_C) was solved at 2.20-Å resolution. Specifically, Lys¹⁸¹ enabled the DNA-binding affinity of EsrB and promoted the in vitro and in vivo pathogenicity of Edwardsiella piscicida. Moreover, EsrB directly regulated the expression of genes associated with basal metabolism, including iron and tricarboxylic acid (TCA) cycles. Furthermore, EsrB enhanced iron transport capability and the enzyme activity of succinate dehydrogenase and pyruvate dehydrogenase in DMEM. Collectively, our structural and ChIP-seq analysis provides valuable insights into the DNA binding mechanism of EsrB which will facilitate our understanding of EsrB coordinating virulence and basal metabolism gene expression.

Importance: As a crucial virulence regulator, EsrB possesses a LuxR-like superfamily domain at the C-terminal, which is conserved within the canonical NarL family regulators. Due to its critically important role in virulence and pathogenicity in fish hosts, the DNA binding ability has been believed to allow EsrB to regulate genes associated with the invasion process of host cells and basal metabolism in response to environmental stimuli. The lack of EsrB's crystal structure has been a major obstacle in understanding the molecular mechanisms of EsrB-DNA interaction which choreographs EsrB-mediated pathogenic behavior. Here, we conducted ChIP-seq and solved the crystal structure of the C-terminal of EsrB (EsrBC) at 2.20-Å resolution, which revealed that EsrB preferred to bind to virulence-associated promoters with a distinct 7'-4-7' pseudopalindromic DNA motif and interacted with metabolic-related promoters with a high AT DNA motif in Dulbecco's Modified Eagle's Medium (DMEM) (mimicking in vivo environments). Our results facilitate a detailed understanding of EsrB's regulatory role in Edwardsiella piscicida pathogenesis and expand our knowledge of virulence regulators in the family Hafniaceae.

The Atacama Desert in Chile is one of the driest and most inhospitable places on Earth. To analyze the diversity and distribution of microbial communities in such an environment, one of the most important and challenging steps is DNA extraction. Using commercial environmental DNA extraction protocols, a mixture of living, dormant, and dead cells of microorganisms is extracted, but separation of the different DNA pools is almost impossible. To overcome this problem, we applied a novel method on soils across a west-east moisture transect in the Atacama Desert to distinguish between extracellular DNA (eDNA) and intracellular DNA (iDNA) at the cell extraction level. Here, we show that a large number of living and potentially active microorganisms, such as Acidimicrobiia, Geodermatophilaceae, Frankiales, and Burkholderiaceae, occur in the hyperarid areas. We observed viable microorganisms involved as pioneers in initial soil formation processes, such as carbon and nitrogen fixation, as well as mineral-weathering processes. In response to various environmental stressors, microbes coexist as generalists or specialists in the desert soil environment. Our results show that specialists compete in a limited range of niches, while generalists tolerate a wider range of environmental conditions. Use of the DNA separation approach can provide new insights into different roles within viable microbial communities, especially in low-biomass environments where RNA-based analyses often fail.IMPORTANCEThe novel e- and iDNA separation technique offers insights into the living community at the cell extraction level in the hyperarid Atacama Desert. This approach provides a new framework for analyzing the composition and structure of the potentially active part of the microbial communities as well as their specialization, ecological network and community assembly process. Our findings underscore the significance of utilizing alternative genomic techniques in low-biomass environments where traditional DNA- and RNA-based analyses may not be feasible. The results demonstrate the viability of the proposed study framework and show that specialized microorganisms are important in initial soil formation processes, including microbial-driven mineral weathering, as well as the fixation of carbon and nitrogen.

Acetic acid is a byproduct of lignocellulose pretreatment and a potent inhibitor of yeast-based fermentation processes. A thicker yeast plasma membrane (PM) is expected to retard the passive diffusion of undissociated acetic acid into the cell. Molecular dynamic simulations suggest that membrane thickness can be increased by elongating glycerophospholipids (GPL) fatty acyl chains. Previously, we successfully engineered Saccharomyces cerevisiae to increase GPL fatty acyl chain length but failed to lower acetic acid net uptake. Here, we tested whether altering the relative abundance of diacylglycerol (DAG) might affect PM permeability to acetic acid in cells with longer GPL acyl chains (DAG^EN). To this end, we expressed diacylglycerol kinase α (DGKα) in DAG^EN. The resulting DAG^EN_Dgkα strain exhibited restored DAG levels, grew in medium containing 13 g/L acetic acid, and accumulated less acetic acid. Acetic acid stress and energy burden were accompanied by increased glucose uptake in DAG^EN_Dgkα cells. Compared to DAG^EN, the relative abundance of several membrane lipids changed in DAG^EN_Dgkα in response to acetic acid stress. We propose that the ability to increase the energy supply and alter membrane lipid composition could compensate for the negative effect of high net acetic acid uptake in DAG^EN_Dgkα under stressful conditions.

Importance: In the present study, we successfully engineered a yeast strain that could grow under high acetic acid stress by regulating its diacylglycerol metabolism. We compared how the plasma membrane and total cell membranes responded to acetic acid by adjusting their lipid content. By combining physiological and lipidomics analyses in cells cultivated in the absence or presence of acetic acid, we found that the capacity of the membrane to adapt lipid composition together with sufficient energy supply influenced membrane properties in response to stress. We suggest that potentiating the intracellular energy system or enhancing lipid transport to destination membranes should be taken into account when designing membrane engineering strategies. The findings highlight new directions for future yeast cell factory engineering.

Wastewater surveillance for infectious agents has proved useful in identifying the circulation of viruses within populations. We investigated the presence and concentration of human immunodeficiency virus (HIV)-1 total nucleic acids (including both viral RNA and proviral DNA) in wastewater solids. We retrospectively measured HIV-1 nucleic acids in two samples per week for 26 months at two wastewater treatment plants serving populations with different prevalences of HIV infections in San Francisco and Santa Clara County, California, USA. We detected HIV nucleic acids in a majority of samples with concentrations ranging from non-detect to 3.9 × 10⁵ cp/g (N = 459 samples total). Concentrations of HIV-1 were significantly higher in samples from the wastewater treatment plant serving a population with a higher prevalence of people living with HIV than in the plant serving a population with a lower prevalence. The HIV-1 nucleic acids amplified were primarily DNA and thus represented proviral DNA shedding into wastewater. Additionally, we found that HIV-1 nucleic acid concentrations in wastewater solids were orders of magnitude higher than those in liquid wastewater indicating that the HIV-1 target preferentially sorbs to solids. Whether concentrations of HIV-1 in wastewater solids can be used to identify the number of incident cases remains unknown. Additional work on HIV-1 shedding from individuals with viremia and people living with HIV is needed to translate wastewater measurements into quantitative information on infections. Additional work may also be needed to document non-human sources of HIV-1 nucleic acids in wastewater.

Importance: Human immunodeficiency virus (HIV)-1 has infected nearly 100 million people since it emerged in the 1980s. Antiretroviral therapy prevents transmission of HIV and also allows infected individuals to live healthy lives with normal life expectancy. Consequently, identifying unrecognized cases of HIV is of paramount importance. Since wastewater represents a composite biological sample from a community, it may provide valuable information on HIV-1 prevalence that can be used to inform HIV testing and outreach.

Biological nitrogen fixation (BNF) is an essential source of new nitrogen (N) for terrestrial ecosystems. The abiotic factors regulating BNF have been extensively studied in various ecosystems and laboratory settings. Despite this, our understanding of the impact of neighboring bacteria on N₂ fixer activity remains limited. Here, we explored this question using a co-culture of the two model species: the free-living diazotroph Azotobacter vinelandii and the non-fixing plant growth-promoting rhizobacteria Bacillus subtilis. We observed that the interaction between the two bacteria was modulated by N availability. Under N-replete conditions, B. subtilis outcompeted A. vinelandii in the co-culture. Under N-limiting conditions, BNF activity by A. vinelandii was enhanced in the presence of B. subtilis. Reciprocally, the presence of A. vinelandii repressed sporulation by B. subtilis and supported its growth likely through N transfer. N inputs by A. vinelandii were doubled in the presence of B. subtilis compared to the monoculture, primarily due to the retention of a robust N₂ fixation activity in the stationary phase. A proteomic analysis revealed that A. vinelandii N metabolism, particularly the molybdenum nitrogenase isoform protein levels (NifK and NifD), was upregulated during the stationary growth phase in the presence of B. subtilis. This study revealed that N stress drives bacterial interactions and activity in a two-species community, especially in the stationary phase.

Importance: Reducing inputs of chemical N fertilizers is essential to develop a more sustainable agriculture. The stimulation of biological nitrogen fixation by N2 fixers in multispecies cultures, here the plant growth-promoting rhizobacteria Azotobacter vinelandii and Bacillus subtilis, opens opportunities for the formulation of biofertilizers consortia. While most research on N2 fixation historically focussed on the exponential growth phase of microorganisms, we observed that Bacillus subtilis stimulated Azotobacter vinelandii N2 fixation mostly during the stationary phase. This result highlights that more research on the factors controlling N2 fixation repression during the stationary growth phase, especially bacteria-bacteria interactions, is eagerly needed.

Freshwater ecosystems can be largely affected by neighboring agriculture fields where potential fertilizer nitrate run-off may leach into surrounding water bodies. To counteract this eutrophic driver, farmers in certain areas are utilizing denitrifying woodchip bioreactors (WBRs) in which a consortium of microorganisms convert the nitrate into nitrogen gases in anoxia, fueled by the degradation of lignocellulose. Polysaccharide-degrading strategies have been well described for various aerobic and anaerobic systems, including the use of carbohydrate-active enzymes, utilization of lytic polysaccharide monooxygenases (LPMOs) and other redox enzymes, as well as the use of cellulosomes and polysaccharide utilization loci (PULs). However, for denitrifying microorganisms, the lignocellulose-degrading strategies remain largely unknown. Here, we have applied a combination of enrichment techniques, gas measurements, multi-omics approaches, and amplicon sequencing of fungal ITS and procaryotic 16S rRNA genes to identify microbial drivers for lignocellulose transformation in woodchip bioreactors and their active enzymes. Our findings highlight a microbial community enriched for (ligno)cellulose-degrading denitrifiers with key players from the taxa Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota). A wide substrate specificity is observed among the many expressed carbohydrate-active enzymes (CAZymes) including PULs from Bacteroidetes. This suggests a broad degradation of lignocellulose subfractions, including enzymes with auxiliary activities whose functionality is still puzzling under strict anaerobic conditions.

Importance: Freshwater ecosystems face significant threats from agricultural runoff, which can lead to eutrophication and subsequent degradation of water quality. One solution to mitigate this issue is using denitrifying woodchip bioreactors (WBRs), where microorganisms convert nitrate into nitrogen gases utilizing lignocellulose as a carbon source. Despite the well-documented polysaccharide-degrading strategies in various systems, the mechanisms employed by denitrifying microorganisms in WBRs remain largely unexplored. This study fills a critical knowledge gap by revealing the degrading strategies of denitrifying microbial communities in WBRs. By integrating state-of-the-art techniques, we have identified key microbial drivers including Giesbergeria, Cellulomonas, Azonexus, and UBA5070 (Fibrobacterota) playing significant roles in lignocellulose transformation and showcasing a broad substrate specificity and complex metabolic capability. Our findings advance the understanding of microbial ecology in WBRs and by revealing the enzymatic activities, this research may inform efforts to improve water quality, protect aquatic ecosystems, and reduce greenhouse gas emissions from WBRs.

Managing water quality with microbial impairment caused by Enterococcus poses unique challenges regarding the determination of fecal host origin. Most water monitoring is performed based on Enterococcus counts that neither detect the location of the introduction of pollution nor identify the type of contaminating Enterococcus. The use of sequenced-based microbial source tracking could allow for identification of fecal origin and potential remediation of pollution. The state of Delaware has numerous waterways with high microbial impairment from unknown sources, so we used sequence-based microbial source tracking to investigate potential microbial pollution in three watersheds with significant variation in land use and population density. In this study, we use a 16S rRNA sequence reference library of microbial communities from relevant fecal sources (wild animal, domestic animal, sediment, and septic/wastewater) to determine the most likely sources of microbial impairment in three Delaware watersheds. This study assigned sources of microbial contamination to mostly human-related sources (septic and wastewater) or unknown sources indicating that waste infrastructure may have a larger influence on microbial community structure in Delaware watersheds than previously considered. Our results suggest that long-term source tracking is valuable for ruling out native or domesticated animals as contributors to water pollution.IMPORTANCETraditional microbial pollution monitoring utilizes specific fecal indicator bacteria that need to grow in the laboratory for detection. Here, we show the use of sequence information from whole microbial communities and an expanded reference library in microbial source tracking. Expanding the host detection range by including the whole microbial community may allow for a wider range of potential fecal origin identification even when specific fecal indicators are absent or in low concentration. We show that many Delaware waterways bear the signature of human influence compared to natural sources. In addition, the robust reference library built in this study can be used to conduct source tracking studies in the mid-Atlantic USA.

Candida albicans is an opportunistic pathogen and colonizer of the human gut and mucosal membranes. C. albicans exhibits morphological plasticity, which is crucial for its fitness within the host and virulence. Morphogenesis in C. albicans is regulated, in part, by its production of farnesol, an autoregulatory molecule that inhibits filamentation. Morphogenesis is also regulated in response to external cues, such as serum, which stimulates hyphal formation by C. albicans. The precise mechanism by which serum stimulates hyphal formation is unknown. The most abundant serum protein is albumin. The binding affinity of albumin for nonpolar, fatty-acid-like molecules suggests that it may interact directly with exogenous farnesol and influence morphogenesis through sequestration of free farnesol. To test this hypothesis, we assessed whether albumin and albumin devoid of fatty acids (i) stimulated farnesol secretion and (ii) influenced the farnesol threshold required to inhibit filamentation. We found that albumin promoted farnesol secretion and filamentation, and the extent of its ability to do so was based on the presence or absence of bound fatty acids. We hypothesize that albumin not bound to fatty acids has the capacity to bind to farnesol and sequester it from C. albicans, encouraging filamentation.IMPORTANCEFor at least 50 years, researchers have wondered about the mechanisms by which serum stimulates germ tube formation (GTF) and hyphal growth in C. albicans. Here, we tested a model (Nickerson et al., Microbiol Mol Biol Rev 88:e00081-22, 2024, https://doi.org/10.1128/mmbr.00081-22) that serum promotes GTF and farnesol synthesis in part by extracting internal farnesol (F_i) from the cells toward the excess binding capacity of the albumins. The data presented here suggests that albumin not bound by fatty acids sequesters free farnesol thereby modulating filamentation and farnesol secretion by altering the equilibrium of internal vs external farnesol. We expect that the influence of secreted farnesol on cell morphology will differ during pathogenesis depending on location within the body, but sequestration of farnesol in the blood could mediate immune cell recruitment and promote hyphal formation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome.

Importance: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.